来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25. years, and lack of auto-antibodies. It accounts for 2-5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil. © 2012 Elsevier B.V.

A population-specific HTR2B stop codon predisposes to severe impulsivity

Impulsivity, describing action without foresight, is an important feature of several psychiatric diseases, suicidality and violent behaviour. The complex origins of impulsivity hinder identification of the genes influencing it and the diseases with which it is associated. Here we perform exon-focused sequencing of impulsive individuals in a founder population, targeting fourteen genes belonging to the serotonin and dopamine domain. A stop codon in HTR2B was identified that is common (minor allele frequency > 1%) but exclusive to Finnish people. Expression of the gene in the human brain was assessed, as well as the molecular functionality of the stop codon, which was associated with psychiatric diseases marked by impulsivity in both population and family-based analyses. Knockout of Htr2b increased impulsive behaviours in mice, indicative of predictive validity. Our study shows the potential for identifying and tracing effects of rare alleles in complex behavioural phenotypes using founder populations, and indicates a role for HTR2B in impulsivity.

Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue

Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Polymorphism in codon 17 of the CTLA-4 gene (+49 A/G) is not associated with susceptibility to rheumatoid arthritis in British Caucasians

The role of the CTLA-4 antigen in the development of autoimmune diseases is well documented, with several autoimmune disorders showing association or linkage with the CTLA-4 locus. Its role in the aetiology of rheumatoid arthritis (RA) however, remains unclear, as the functional studies of the B7-CTLA-4 pathway in mouse models of RA and genetic studies in humans have given contrasting results. We have studied the single nucleotide polymorphism at position +49 (A/G) of the CTLA-4 gene, in a cohort of 421 RA cases and 452 healthy controls from the UK. Despite the high statistical power to detect even a weak susceptibility effect, no significant association was found. We also analysed the distribution of the allele and genotype frequencies with respect to the presence of the shared epitope (a known RA susceptibility factor) and found no statistically significant differences. We conclude that, although the importance of the B7-CTLA-4 interaction in the development of RA can not be excluded, the CTLA-4 gene is unlikely to be a predisposing factor to this disease.

Communication pathways from the anti-codon region to the aminoacylation site in Methionyl tRNA Synthetase: Molecular dynamics simulations and the structure network analysis

The enzymes of the family of tRNA synthetases perform their functions with high precision by synchronously recognizing the anticodon region and the aminoacylation region, which are separated by ?70 in space. This precision in function is brought about by establishing good communication paths between the two regions. We have modeled the structure of the complex consisting of Escherichia coli methionyl-tRNA synthetase (MetRS), tRNA, and the activated methionine. Molecular dynamics simulations have been performed on the modeled structure to obtain the equilibrated structure of the complex and the cross-correlations between the residues in MetRS have been evaluated. Furthermore, the network analysis on these simulated structures has been carried out to elucidate the paths of communication between the activation site and the anticodon recognition site. This study has provided the detailed paths of communication, which are consistent with experimental results. Similar studies also have been carried out on the complexes (MetRS + activated methonine) and (MetRS + tRNA) along with ligand-free native enzyme. A comparison of the paths derived from the four simulations clearly has shown that the communication path is strongly correlated and unique to the enzyme complex, which is bound to both the tRNA and the activated methionine. The details of the method of our investigation and the biological implications of the results are presented in this article. The method developed here also could be used to investigate any protein system where the function takes place through long-distance communication.

WebGeSTer DB-a transcription terminator database

We present WebGeSTer DB, the largest database of intrinsic transcription terminators (http://pallab.serc.iisc.ernet.in/gester). The database comprises of a million terminators identified in 1060 bacterial genome sequences and 798 plasmids. Users can obtain both graphic and tabular results on putative terminators based on default or user-defined parameters. The results are arranged in different tiers to facilitate retrieval, as per the specific requirements. An interactive map has been incorporated to visualize the distribution of terminators across the whole genome. Analysis of the results, both at the whole-genome level and with respect to terminators downstream of specific genes, offers insight into the prevalence of canonical and non-canonical terminators across different phyla. The data in the database reinforce the paradigm that intrinsic termination is a conserved and efficient regulatory mechanism in bacteria. Our database is freely accessible.

Functional Significance of an Evolutionarily Conserved Alanine (GCA) Resume Codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Phylogeographic analysis of mitochondrial DNA haplogroup F2 in China reveals T12338C in the initiation codon of the NDS gene not to be pathogenic

In this report, we studied on a homoplasmic T12338C change in mitochondrial DNA (mtDNA), which substituted methionine in the translational initiation codon of the NADH dehydrogenase subunit 5 gene (ND5) with threonine. This nucleotide change was originall

Evidence of gene conversion in the evolutionary process of the codon 41/42 (-CTTT) mutation causing beta-thalassemia in southern China

The 4-bp deletion (-CTTT) at codon 41/42 (CD41/42) of the human beta-globin gene represents one of the most common beta-thalassemia mutations in East Asia and Southeast Asia, which is historically afflicted with endemic malaria, thus hypothetically evolvi

Is there a close relationship between synonymous codon bias and codon-anticodon binding strength in human genes?

Synonymous codon bias has been examined in 78 human genes (19967 codons) and measured by relative synonymous codon usage (RSCU). Relative frequencies of all kinds of dinucleotides in 2,3 or 3,4 codon positions have been calculated, and codon-anticodon bin

The features of synonymous codon bias and GC-content relationship in human genes

728 human genes were divided to four groups according to the GC contents of their coding sequences (from GC<0.43 to GC>0.58). Examination of synonymous-codon bias in the 4 groups show that NTG (N represents any base of T, A, C, G) is most favored and NCG